High Glucose-induced VSMC proliferation and migration is associated with decreased PGC-1α expression.

<p>Cultured rat VSMCs were incubated for 48 h in 5.5 mM, 11 mM, 15 mM and 25 mM glucose respectively, Cell growth was determined by counting the number of cells (A) and VSMC migration was determined by a standard wound healing assay (B), PGC-1α expression was determined by real-time RT-PCR by using primers specific for rat PGC-1α and β-actin (C). The bars represent means±S.E.M (n = 6). *P<0.05, **P<0.01, #P<0.001 vs. cells incubated in 5.5 mM glucose. Arterial samples were obtained from the normal and STZ injected rats, the intima and outer and inner tissue layers were removed from arteries. PGC-1α expression was determined by real-time RT-PCR. Data (n = 10) were expressed as means±S.E.M. *P<0.05 vs. normal rats (D).</p

Suppression of PGC-1α abolishes the inhibitory effect of palmitic acid on high glucose-induced VSMC growth and movement.

<p>VSMCs were transfected with siRNA (SI) or the negative control (N), then left stimulated with 15 mM gluoose (HG) in the presence of 0.4 mM palmitic acid (PA) for 48 h. Cell growth was determined by counting the number of cells (A). Migration distance was determined by a standard wound healing assay (B) and by transwell analysis (C). The bars represent means±S.E.M (n = 6). *P<0.05, **P<0.01, #P<0.001 compared with the N+PA+HG group.</p

Knock-down of PGC-1α accelerated high glucose-induced VSMC proliferation and migration.

<p>VSMCs transfected with siRNA (SI) or the negative control (N), were either left untreated or stimulated with 15 mM gluoose (HG). The interference effect was assessed by quantitative PCR analysis and data were shown as the ratio of PGC-1α/β-actin (A), **P<0.01compared with the negative control group (N). 120 µg total proteins from VSMCs were assayed for Western blot with PGC-1 antibody to confirm the interference effiency (B). Effects of decreased PGC-1α on high glucose-induced VSMC proliferation were determined by cell counting (C). VSMC migration was determined by wound healing (D) and transwell analysis (E). Individual data points in this figure represents the mean±S.E.M (n = 6). *P<0.05, #P<0.001 compared with the control group (N+HG).</p

Increase of PGC-1α either exogenously by adenovirus or endogenously by palmitic acid abolished high glucose induced phosphorylation of ERK1/2 in VSMCs.

<p>VSMCs were infected with adenovirus for 24 h, then made quiescent by serum-starvation for 6 h, and then stimulated with HG for 4 h. Total protein were harvested and used to detect ERK1/2 activity (A). Cultured VSMCs in 5.5 mM glucose were made quiescent by serum-starvation for 6 h, then cells were either left untreated or stimulated with 15 mM glucose (HG) in the absence and presence of 0.4 mM palmitic acid (PA) for 12 h. ERK activity were determined by Western blotting with ERK1/2 (pT202/pY204) Phospho-Specific antibodies compared with b-tubulin antibody and densitometric analysis of ERK1/2 activity are showed. Representative blots of three similar results are showed. #P<0.001 compared with control.</p

Palmitic acid stimulates PGC-1α expression and retards high glucose-induced VSMC growth and movement.

<p>Cultured rat VSMCs in 5.5 mM glucose were either left untreated or stimulated with 15 mM glucose (HG) in the absence and presence of 0.4 mM palmitic acid (PA) for 48 h. PGC-1α expression was determined by real-time RT-PCR (A) and by Western blot with PGC-1 antibody compared with GAPDH antibody, 120 µg total proteins from VSMCs were loaded (B). Cell growth was determined by counting the number of cells (C). Migration distance was determined by a standard wound healing assay (D) and transwell analysis (E). The bars represent means±S.E.M (n = 6). **P<0.01 compared with control conditions, #P<0.001 compared with the control group.</p

Overexpression of PGC-1α suppresses high glucose induced VSMC proliferation and migration.

<p>Cultured rat VSMCs in 5.5 mM glucose infected with adenovirus driving the expression of PGC-1α (PGC group) or GFP (GFP group) were either left untreated or incubated with 15 mM gluoose (HG) for 48 h. PGC-1α mRNA expression was determined by real-time RT-PCR (A), the values of PGC-1α/β-actin were normalized to that of control. 60 µg total proteins from VSMCs were assayed for Westernblot with PGC-1 antibody (B). Cell growth was determined by counting the number of cells (C). VSMC migration was determined by a standard wound healing assay (D) and by transwell analysis (E). The bars represent means±S.E.M (n = 6). **P<0.01, #P<0.001 compared with the GFP control group (GFP group).</p